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Research
Modulation of the Permeability-Inducing Factor Angiopoietin-2 Through Bifonazole in Systemic Inflammation
Sascha David
Hannover Medical School Department of Nephrology and Hypertension and Institute for Intensive Care, University Hospital Zurich
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8231-0461
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Sepsis is a life-threatening organ dysfunction due to a pathological host response to an infection. Vascular barrier breakdown represents a key component of the maladaptive host response and the release of pre-stored endothelial Angiopoietin-2 (Angpt-2) is a direct driver of endothelial hyperpermeability. Although it has been demonstrated that Angpt-2 is associated with morbidity and mortality, a therapeutic approach targeting this injurious endothelial protein is not available. We screened for FDA approved drugs that might have off-target effects decreasing circulating Angpt-2 levels and therefore, ameliorating capillary leakage.
Methods
Endothelial cells were isolated from human umbilical veins (HUVECs) and used for in vitro studies at baseline and after stimulation (FDA-library screening, RT-PCR, ELISA, immunocytochemistry). On the functional level, we assessed real-time transendothelial electrical resistance (TER) using the ECIS (electric cell-substrate impedance sensing) device.
Results
We identified Bifonazole (BIFO) in an unbiased library screen and found that it is able to reduce spontaneous Angpt-2 release in HUVECs in a time- and dose-dependent manner after 8, 12 and 24 h (24 h: veh: 15.6 ± 0.7 vs. BIFO: 8.6 ± 0.8 ng/mL, p < 0.0001). BIFO reduced not only Angpt-2 release but also its intra-cellular content by 33 % (p < 0.001). Stimulation of HUVECs with a sepsis mediator, i.e. tumor necrosis factor α (TNFα) (10 ng/ml) induced a rapid release of Angpt-2 that could analogously be blocked by additional treatment with BIFO (veh: 1.58 ± 0.2 vs. BIFO: 1.02 ± 0.1, p < 0.0001). On the functional level, the quantification of endothelial permeability by TER revealed that BIFO was sufficient to reduce an acute barrier breakdown induced by Thrombin (veh: 0.82 ± 0.1 vs. BIFO: 1.01 ± 0.02, p < 0.05).
Conclusion
The antifungal BIFO can reduce both release and biosynthesis of the endothelial-destabilizing factor Angpt-2 in vitro. Henceforth, our results suggest that BIFO might counterpart the pathophysiology of endothelial permeability in systemic inflammation. Additional studies are needed to further investigate the underlying mechanism and to translate these findings to in vivo models.
Keywords
Angiopoietin-2, Bifonazole, Sepsis, systemic inflammation
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Research
Modulation of the Permeability-Inducing Factor Angiopoietin-2 Through Bifonazole in Systemic Inflammation
Sascha David
Hannover Medical School Department of Nephrology and Hypertension and Institute for Intensive Care, University Hospital Zurich
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8231-0461
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 22 Sep, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Critical Care & Emergency Medicine
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Sepsis is a life-threatening organ dysfunction due to a pathological host response to an infection. Vascular barrier breakdown represents a key component of the maladaptive host response and the release of pre-stored endothelial Angiopoietin-2 (Angpt-2) is a direct driver of endothelial hyperpermeability. Although it has been demonstrated that Angpt-2 is associated with morbidity and mortality, a therapeutic approach targeting this injurious endothelial protein is not available. We screened for FDA approved drugs that might have off-target effects decreasing circulating Angpt-2 levels and therefore, ameliorating capillary leakage.
Methods
Endothelial cells were isolated from human umbilical veins (HUVECs) and used for in vitro studies at baseline and after stimulation (FDA-library screening, RT-PCR, ELISA, immunocytochemistry). On the functional level, we assessed real-time transendothelial electrical resistance (TER) using the ECIS (electric cell-substrate impedance sensing) device.
Results
We identified Bifonazole (BIFO) in an unbiased library screen and found that it is able to reduce spontaneous Angpt-2 release in HUVECs in a time- and dose-dependent manner after 8, 12 and 24 h (24 h: veh: 15.6 ± 0.7 vs. BIFO: 8.6 ± 0.8 ng/mL, p < 0.0001). BIFO reduced not only Angpt-2 release but also its intra-cellular content by 33 % (p < 0.001). Stimulation of HUVECs with a sepsis mediator, i.e. tumor necrosis factor α (TNFα) (10 ng/ml) induced a rapid release of Angpt-2 that could analogously be blocked by additional treatment with BIFO (veh: 1.58 ± 0.2 vs. BIFO: 1.02 ± 0.1, p < 0.0001). On the functional level, the quantification of endothelial permeability by TER revealed that BIFO was sufficient to reduce an acute barrier breakdown induced by Thrombin (veh: 0.82 ± 0.1 vs. BIFO: 1.01 ± 0.02, p < 0.05).
Conclusion
The antifungal BIFO can reduce both release and biosynthesis of the endothelial-destabilizing factor Angpt-2 in vitro. Henceforth, our results suggest that BIFO might counterpart the pathophysiology of endothelial permeability in systemic inflammation. Additional studies are needed to further investigate the underlying mechanism and to translate these findings to in vivo models.
Figure 1
Figure 2
Figure 3
Figure 4